Method for detecting characteristic chromatogram of Jiaojianshengqu formula granules and quality control method

Abstract

The present application relates to the technical field of traditional Chinese medicine detection, and provide a kind of characteristic spectrum detection method and quality control method of Jiajian Shenqu formula granules, using high performance liquid chromatography, chromatographic conditions are as follows: with octadecylsilane bonded silica as filler;With methanol as mobile phase A, with 0.2% phosphoric acid solution as mobile phase B;Gradient elution program is as follows: 0-15min, the volume ratio of mobile phase A and mobile phase B is 0%:100%;15-50min, the volume ratio of mobile phase A and mobile phase B is 0%:5%→100%:95%;50-60min, the volume ratio of mobile phase A and mobile phase B is 5%:95%→8%:92%;60-65min, the volume ratio of mobile phase A and mobile phase B is 8%:92%;The above method can comprehensively, quickly detect the effective component and its content in Jiajian Shenqu formula granules.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and specifically relates to a detection method and a quality control method for the characteristic fingerprint of Jiaojianshenqu formula granules. Background Art

[0002] Jianshenqu is a processed product made by mixing fine powders of drugs such as Pogostemonis Herba, Artemisiae Annuae Herba, etc. with flour and fermenting. It has a pungent, sweet taste and a warm nature. It belongs to the spleen and stomach meridians. It has the effects of promoting digestion and resolving food stagnation, strengthening the spleen and regulating the stomach. It is commonly used for symptoms such as retained food stagnation, fullness and distension in the chest and abdomen, and spleen deficiency diarrhea. Jiaojianshenqu is the stir-fried product of Jianshenqu, and after frying, its function of promoting digestion and resolving food stagnation, strengthening the spleen and regulating the stomach can be enhanced. Jiaojianshenqu contains components such as fatty oil, sugars, yeast, etc.

[0003] The quality control of Jiaojianshenqu in local standards includes items such as original plant variety, cut crude drug processing, cut crude drug properties, physicochemical identification, etc. The literature also elaborates on the chemical components in Jiaojianshenqu, including components such as fatty oil, sugars, yeast, etc. However, the detection standards for Jiaojianshenqu only involve simple physicochemical identifications such as moisture and extractives, and do not detect all components comprehensively. At present, the quality control of Jiaojianshenqu is all针对焦建神曲饮片,并非是针对焦建神曲的药物制剂。虽然焦建神曲配方颗粒系由焦建神曲饮片经过生产加工成的一种制剂类型,但经过生产加工处理的焦建神曲配方颗粒相比焦建神曲饮片,理化性质发生了很大的改变,因而,针对焦建神曲饮片的检测方法并不能适用于焦建神曲的药物制剂。 Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a detection method and a quality control method for the characteristic fingerprint of Jiaojianshenqu formula granules, which can be more specific and targeted for Jiaojianshenqu formula granules, play a role in standardized, normalized and objective description and evaluation, can comprehensively and rapidly detect Jiaojianshenqu formula granules, and is of great significance for its comprehensive quality detection and overall quality control.

[0005] For this purpose, the present invention provides the following technical solutions:

[0006] A detection method for the characteristic fingerprint of Jiaojianshenqu formula granules, which is detected by high performance liquid chromatography, and the chromatographic conditions are:

[0007] Using octadecylsilane chemically bonded silica as the filler; using methanol as mobile phase A and 0.2 wt% phosphoric acid solution as mobile phase B; the gradient elution program is:

[0008] 0 - 15 min, the volume ratio of mobile phase A to mobile phase B is 0%:100%;

[0009] Over 15-50 minutes, the volume ratio of mobile phase A to mobile phase B changes from 0%:5% to 100%:95%.

[0010] Over 50-60 minutes, the volume ratio of mobile phase A to mobile phase B changes from 5%:95% to 8%:92%.

[0011] The volume ratio of mobile phase A to mobile phase B is 8%:92% for 60-65 minutes.

[0012] Optionally, the chromatographic conditions include at least one of the following:

[0013] 1) Detection wavelength: 262nm;

[0014] 2) The flow rate is 0.75-0.85 mL / min;

[0015] 3) Column temperature is 23-27℃;

[0016] 4) The chromatographic column has the following specifications: column length of 250 mm, inner diameter of 4.6 mm, and particle size of 5 μm; optionally, the chromatographic column is Inertil ODS-3, Agilent SB-C18, or Waters HSS T3.

[0017] 5) The injection volume is 5-15 μL, with 10 μL being an option.

[0018] Optionally, it also includes the preparation of the test solution, including: taking the test sample, adding solvent to extract, filtering, and taking the filtrate to obtain the solution;

[0019] Optionally, the solvent is water;

[0020] Optionally, the mass-volume ratio of the test sample to the solvent is (0.25-1):10, preferably 0.5:10, with a ratio of g / ml.

[0021] Optionally, the preparation of a reference solution for the control medicinal slices is also included: take Jiaojian Shenqu control medicinal slices, add solvent to extract, filter, and take the filtrate to obtain the solution;

[0022] Optionally, the solvent is water;

[0023] Optionally, the mass-to-volume ratio of the Jiao Jian Shen Qu reference herbal slices to the solvent is (0.5-2):10-40, preferably 1:20, with a ratio of g / ml;

[0024] Optionally, take 0.5-2 parts by weight of Jiaojian Shenqu reference slices and add them to 10-40 parts by volume of water. Heat and reflux to extract for 0.75-1.5 hours, filter, evaporate the filtrate to dryness, add the residue to 5-10 parts by volume of water, sonicate, filter, and collect the filtrate.

[0025] Optionally, the preparation of a reference solution may also be included: take an appropriate amount of uridine reference standard, accurately weigh it, and add water to prepare a solution containing 10-100 μg per ml. Preferably, take an appropriate amount of uridine reference standard, accurately weigh it, and add water to prepare a solution containing 50 μg per ml.

[0026] A method for detecting the content of Jiaojian Shenqu formula granules includes detection by high performance liquid chromatography (HPLC) according to the method for detecting the characteristic chromatogram of Jiaojian Shenqu formula granules.

[0027] The application of the method for detecting the characteristic spectrum of Jiaojian Shenqu formula granules or the method for determining the content of Jiaojian Shenqu formula granules in the quality testing of Jiaojian Shenqu formula granules.

[0028] A method for quality testing of Jiaojian Shenqu formula granules includes the steps of obtaining a characteristic spectrum of the test sample according to the method for detecting the characteristic spectrum of Jiaojian Shenqu formula granules, and comparing the characteristic spectrum with a reference characteristic spectrum.

[0029] The control feature spectrum is obtained by fitting the feature spectrum obtained by using at least one batch of Jiaojian Shenqu formula granules as a standard according to the detection method of the feature spectrum of Jiaojian Shenqu formula granules.

[0030] Optionally, the reference characteristic spectrum includes 15 characteristic peaks, with peak 9 corresponding to uridine; peak 9 is used as the reference peak S, and the relative retention times of peaks 1-8 and peaks 10-15 should be within ±10% of the specified values. The specified values ​​for peak 1 are 0.16, peak 2 is 0.18, peak 3 is 0.21, peak 4 is 0.29, peak 5 is 0.34, peak 6 is 0.39, peak 7 is 0.44, peak 8 is 0.84, peak 10 is 1.08, peak 11 is 1.27, peak 12 is 1.43, peak 13 is 1.55, peak 14 is 1.87, and peak 15 is 2.26.

[0031] Optionally, peak 4 corresponds to adenine, peak 7 corresponds to hypoxanthine, peak 8 corresponds to xanthine, peak 9 (S) corresponds to uridine, and peak 10 corresponds to thymine.

[0032] The technical solution of this invention has the following advantages:

[0033] 1. This invention provides a method for detecting the characteristic chromatogram of Jiaojian Shenqu formula granules, using high-performance liquid chromatography (HPLC). The chromatographic conditions are as follows: octadecylsilane-bonded silica gel is used as the packing material; methanol is used as mobile phase A, and 0.2 wt% phosphoric acid solution is used as mobile phase B; the gradient elution program is as follows: 0-15 min, the volume ratio of mobile phase A to mobile phase B is 0%:100%; 15-50 min, the volume ratio of mobile phase A to mobile phase B is 0%:5% → 100%:95%; 50-60 min... For the first 60-65 minutes, the volume ratio of mobile phase A to mobile phase B is 5%:95% → 8%:92%; for the second 60-65 minutes, the volume ratio of mobile phase A to mobile phase B is 8%:92%. This method is specifically designed for the detection of Jiaojian Shenqu formula granules, with good separation. It can simultaneously determine the content of uridine, hypoxanthine, adenine, thymine, and xanthine. Moreover, this detection method has high precision, good stability, and good repeatability. Therefore, it can comprehensively and rapidly detect the effective components and their content in Jiaojian Shenqu formula granules.

[0034] Furthermore, the detection method provided by this invention can select S-peak uridine as the internal reference peak in the fingerprint spectrum, and can determine 15 common characteristic peaks of Jiaojian Shenqu formula granules. The relative retention time of each common characteristic peak can be calculated based on S-peak uridine, which is beneficial for comprehensive quality detection and overall quality control of Jiaojian Shenqu formula granules, thereby helping to improve the safety and stability of the drug. Attached Figure Description

[0035] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 These are characteristic chromatograms of 18 batches of Jiaojian Shenqu formula granules in Example 1 of this invention;

[0037] Figure 2 This is a comparative characteristic spectrum of the Jiaojian Shenqu formula granules in Example 1 of the present invention;

[0038] Figure 3 This is a characteristic chromatogram of the Jiao Jian Shen Qu (a type of medicinal herb) control slices in Example 1 of this invention;

[0039] Figure 4 This is a positioning diagram of the reference standard of Jiaojian Shenqu formula granules in Example 1 of the present invention;

[0040] Figure 5This is a characteristic chromatogram of the Jiaojian Shenqu formula granules with a flow rate of 0.75 ml / min in Example 2 of the present invention;

[0041] Figure 6 This is a characteristic chromatogram of the Jiaojian Shenqu formula granules with a flow rate of 0.80 ml / min in Example 2 of the present invention;

[0042] Figure 7 This is a characteristic chromatogram of the Jiaojian Shenqu formula granules with a flow rate of 0.85 ml / min in Example 2 of the present invention;

[0043] Figure 8 This is a characteristic spectrum of the Jiaojian Shenqu formula granules with a column temperature of 23°C in Example 3 of the present invention;

[0044] Figure 9 This is a characteristic spectrum of the Jiaojian Shenqu formula granules with a column temperature of 25°C in Example 3 of the present invention;

[0045] Figure 10 This is a characteristic spectrum of the Jiaojian Shenqu formula granules with a column temperature of 27°C in Example 3 of the present invention;

[0046] Figure 11 This is a characteristic chromatogram of the Jiaojian Shenqu formula granules with an Inertil ODS-3 chromatographic column in Example 4 of the present invention;

[0047] Figure 12 This is a characteristic chromatogram of the Jiaojian Shenqu formula granules in Example 4 of the present invention, with an Agilent SB-C18 chromatographic column;

[0048] Figure 13 This is a characteristic chromatogram of the Jiaojian Shenqu formula granules in Example 4 of the present invention, with a Waters HSS T3 chromatographic column.

[0049] Figure 14 This is the negative blank control chromatogram in the specificity experiment of Experiment Example 1 of this invention;

[0050] Figure 15 This is the chromatogram of the uridine reference standard in the specificity experiment of Experiment Example 1 of this invention;

[0051] Figure 16 This is the chromatogram of the reference decoction piece of Jiaojian Shenqu in the specificity experiment of Experiment Example 1 of this invention;

[0052] Figure 17 This is the chromatogram of the Jiaojian Shenqu formula granules test sample in the specificity experiment of Experiment Example 1 of this invention. Detailed Implementation

[0053] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0054] Where specific experimental steps or conditions are not specified in the examples, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0055] The pharmaceutical preparation of Jiaojian Shenqu described in this invention is prepared by the following method:

[0056] Take Jiaojian Shenqu (a type of medicinal fermented wheat), heat and reflux extract at least once, adding 6-12 times its weight of water each time for at least 0.5 hours. Filter, combine the filtrates, and concentrate the filtrate to a relative density of 1.05-1.10 g / mL at 60℃. Add conventional excipients and follow conventional processes to prepare clinically acceptable tablets, capsules, pills, granules, honey-refined pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, or injectable preparations. The pharmaceutically acceptable excipients include: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrix, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silica, etc.; suspending agents include: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch paste, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various flavorings; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorethidium acetate, eucalyptus oil, etc.; matrix includes: PEG6000, PEG4000, insect wax, etc.

[0057] The test sample used in this embodiment is Jiaojian Shenqu formula granules. The specific preparation method of Jiaojian Shenqu formula granules is as follows: Jiaojian Shenqu is taken and extracted twice by heating and reflux. For the first extraction, 10 times the weight of water is added and soaked for 30 minutes, and then heated and refluxed for 0.5 hours. After filtration, 8 times the weight of water is added and extracted for 0.5 hours. After filtration, the filtrates are combined and concentrated to a relative density of 1.05 g / mL at 60°C. The filtrates are then spray-dried, and the dry powder is mixed with the excipient maltodextrin. After mixing evenly, the mixture is dry-granulated to produce granules.

[0058] Chromatograph 1: Thermo Fisher U3000 chromatography system, including LPG-3400A quaternary pump, WPS-3000TSL autosampler, PDA diode array detector, and chromatography workstation;

[0059] Chromatograph 2: Waters Arc chromatography system, including a quaternary gradient pump, a 120-position high-performance autosampler, an original imported column oven, a Waters 2998 diode array UV detector, and an Empower chromatography management system;

[0060] Chromatographic column:

[0061] Waters XSelect HSS T3 (4.6mm×250mm, 5μm);

[0062] Agilent ZORBAX SB-C18 (4.6mm×250mm, 5μm);

[0063] GL Sciences Inertsil ODS-3 (4.6mm×250mm, 5μm);

[0064] Table 1

[0065]

[0066] Reagents:

[0067] Methanol and acetonitrile were chromatographic grade (Merck); phosphoric acid (chromatographic grade, 85-90%, Aladdin); glacial acetic acid (HPLC, >99.9%, Aladdin); formic acid (chromatographic grade, ≥98%, Aladdin); water (Milli-Q).

[0068] Drug trials:

[0069] Jiao Jian Shen Qu (medicated leaven) control slices (purchased from Shanghai Hongyong Biotechnology Co., Ltd., batch number 290134-202206).

[0070] Uric acid reference standard (purchased from the National Institutes for Food and Drug Control, batch number 110887-202104, purity 99.6%);

[0071] Uric acid reference standard (purchased from the National Institutes for Food and Drug Control, batch number 110887-202305, purity 99.6%);

[0072] Jiaojian Shenqu Formula Granules (Batch Nos.: 2002003S, 2004001S, 2000403W, 2007003S, 2008001W, 2011002S, 2011003S, 2102001W, 2102002W, 2104002S, 2110001W, 2112002S, 2203001W, 2204002W, 2206003W, 2208002W, 2301002S, 2307003S).

[0073] Example 1

[0074] This embodiment provides a method for detecting the fingerprint spectrum of a pharmaceutical preparation of Jiao Jian Shen Qu, including:

[0075] (1) Preparation of test solution: Take about 0.5g of Jiao Jian Shen Qu drug preparation powder, accurately weigh it, put it in a stoppered conical flask, accurately add 10ml of water, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, add water to make up the lost weight, shake well, filter it, and take the filtrate to obtain the test solution.

[0076] Preparation of reference solution: Take 1g of Jiaojian Shenqu reference slices, place them in a stoppered conical flask, add 20ml of water, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 5ml of water to the residue, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution for the reference slices. Take an appropriate amount of uridine reference standard, accurately weigh it, add water to prepare a solution containing 50μg per ml, which is the reference solution for the standard.

[0077] (2) High-performance liquid chromatography (HPLC) analysis conditions

[0078] Chromatographic column: Octadecylsilane-bonded silica gel was used as the packing material (Inertil ODS-3 column, 250 mm length, 4.6 mm inner diameter, 5 μm particle size); methanol was used as mobile phase A, and 0.2 wt% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; detection wavelength was 262 nm; column temperature was 25℃; flow rate was 0.8 mL / min. The theoretical plate number, calculated based on the uridine peak, should be no less than 3000.

[0079] Table 2. Chromatographic conditions

[0080] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～15 0 100 15～50 0→5 100→95 50～60 5→8 95→92 60～65 8 92

[0081] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0082] In this embodiment, 18 batches of Jiaojian Shenqu formula granules samples were taken as the test solution, and the characteristic fingerprint of Jiaojian Shenqu formula granules was obtained by high performance liquid chromatography, as Figure 1 shown and in the following table. The results showed that the relative retention time values of each characteristic peak of the 18 batches of Jiaojian Shenqu formula granules were within ±10% of the specified values. Among them, S1 to S18 are the batch numbers S1 to S18 in sequence: 2002003S, 2004001S, 2000403W, 2007003S, 2008001W, 2011002S, 2011003S, 2102001W, 2102002W, 2104002S, 2110001W, 2112002S, 2203001W, 2204002W, 2206003W, 2208002W, 2301002S, 2307003S. Figure 1

[0083] Using the fingerprint similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission, the reference characteristic fingerprint of Jiaojian Shenqu formula granules was generated as Figure 2 shown. There were 15 common chromatographic peaks in the HPLC characteristic fingerprint of Jiaojian Shenqu formula granules obtained. By consulting the literature, the chemical properties of each characteristic peak of Jiaojian Shenqu formula granules were analyzed, and uridine was selected for its localization and identification. Combining with the characteristic fingerprint of the reference cut crude drug, the results are as Figures 3-4 (In the figure, S1 is the reference characteristic fingerprint of Jiaojian Shenqu, S2 is adenine reference, S3 is hypoxanthine reference, S4 is uridine reference, S5 is thymine reference, S6 is xanthine reference) shown and in the following table. It can be seen that there are 15 characteristic peaks in the reference characteristic fingerprint, which correspond to the retention times of 15 common peaks in the reference chromatogram of the reference crude drug. Among them, peak 9 should correspond to the retention time of the uridine reference substance peak; the peak corresponding to the uridine reference substance peak is the S peak. Calculate the relative retention times of peaks 1 - 8, peaks 10 - 15, and their relative retention times should be within the range of ±10% of the specified values. The specified values are: 0.16 (peak 1), 0.18 (peak 2), 0.21 (peak 3), 0.29 (peak 4), 0.34 (peak 5), 0.39 (peak 6), 0.44 (peak 7), 0.84 (peak 8), 1.08 (peak 10), 1.27 (peak 11), 1.43 (peak 12), 1.55 (peak 13), 1.87 (peak 14), 2.26 (peak 15). Peak 4 corresponds to adenine, peak 7 corresponds to hypoxanthine, peak 8 corresponds to xanthine, peak 9 (S) corresponds to uridine, and peak 10 corresponds to thymine.

[0084] Table 3. Determination results of the characteristic fingerprints of 18 batches of Jiaojian Shenqu formula granules

[0085]

[0086]

[0087]

[0088] Table 4. Similarity results of the characteristic spectral maps of 18 batches of Jiaojian Shenqu formula granules

[0089] batch number Similarity batch number Similarity R 1.000 R 1.000 2002003S 0.910 2104002S 0.959 2004001S 0.908 2110001W 0.985 2000403W 0.900 2112002S 0.938 2007003S 0.945 2203001W 0.948 2008001W 0.922 2204002W 0.935 2011002S 0.917 2206003W 0.935 2011003S 0.955 2208002W 0.936 2102001W 0.938 2301002S 0.911 2102002W 0.924 2307003S 0.907

[0090] Table 5. Relative retention time of common patterns in Jiao Jian Shen Qu formula granules

[0091]

[0092] Table 6. Common Pattern Matching Data for Jiao Jian Shen Qu Formula Granules

[0093]

[0094]

[0095] Table 7. Relative retention time of characteristic chromatograms of Jiao Jian Shen Qu (a type of medicinal herb) compared to other medicinal slices.

[0096]

[0097] The results above indicate that the characteristic chromatogram of the test sample, Jiaojian Shenqu granules, should exhibit 15 characteristic peaks, corresponding to the retention times of the 15 common peaks in the chromatogram of the reference medicinal material. Peak 9 should correspond to the retention time of the uridine reference peak. The peak corresponding to the uridine reference peak is the S peak. The relative retention times of peaks 1-8 and 10-15 should be calculated and should be within ±10% of the specified values. The specified values ​​are: 0.16 (peak 1), 0.18 (peak 2), 0.21 (peak 3), 0.29 (peak 4), 0.34 (peak 5), 0.39 (peak 6), 0.44 (peak 7), 0.84 (peak 8), 1.08 (peak 10), 1.27 (peak 11), 1.43 (peak 12), 1.55 (peak 13), 1.87 (peak 14), and 2.26 (peak 15). Peak 4 corresponds to adenine, peak 7 corresponds to hypoxanthine, peak 8 corresponds to xanthine, peak 9 (S) corresponds to uridine, and peak 10 corresponds to thymine.

[0098] Furthermore, when using the characteristic chromatogram detection method of the Jiao Jian Shen Qu pharmaceutical preparation of this embodiment for quality control, the test sample is detected according to the above method, and the obtained chromatogram of the test sample is compared with... Figure 2 The comparative analysis of the reference characteristic chromatograms shown is used for the quality control of the formulation granules. The specific method for quality control using reference characteristic fingerprint chromatograms is as follows:

[0099] pass Figure 2It is known that the HPLC characteristic chromatogram of the test sample, Jiaojian Shenqu formula granules, has 15 common chromatographic peaks, which should correspond to the retention times of the 15 common peaks in the chromatogram of the reference medicinal material. Among them, peak 9 should correspond to the retention time of the uridine reference peak. The peak corresponding to the uridine reference peak is the S peak. The relative retention times of peaks 1-8 and peaks 10-15 should be calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.16 (peak 1), 0.18 (peak 2), 0.21 (peak 3), 0.29 (peak 4), 0.34 (peak 5), 0.39 (peak 6), 0.44 (peak 7), 0.84 (peak 8), 1.08 (peak 10), 1.27 (peak 11), 1.43 (peak 12), 1.55 (peak 13), 1.87 (peak 14), and 2.26 (peak 15).

[0100] The detection method described in this embodiment can effectively obtain fingerprint spectra with good separation of each characteristic peak, and can also simultaneously determine the content of uridine, hypoxanthine, adenine, thymine, and xanthine. Furthermore, by selecting the content of uridine at peak S as the internal reference peak in the fingerprint spectrum, the relative retention times of the common characteristic peaks 1-15 of Jiao Jian Shen Qu formula granules can be determined. Therefore, it can comprehensively and rapidly detect Jiao Jian Shen Qu formula granules, which is beneficial for the comprehensive quality testing and overall quality control of Jiao Jian Shen Qu formula granules, thereby helping to improve the safety and stability of the drug.

[0101] Example 2: Investigation of Flow Rate

[0102] The difference between this embodiment and Example 1 is that the characteristic chromatograms of Jiaojian Shenqu formula granules (2307003S) were detected at different flow rates in the chromatographic conditions. The flow rates were set to 0.80 ml / min, 0.85 ml / min, and 0.75 ml / min, respectively. The results are as follows: Figure 5-7 The table below shows that with slight fluctuations in flow rate, the separation effect of each chromatographic peak is good and the system adaptability parameters are relatively close. However, the relative retention time of each characteristic peak shows significant fluctuations. Considering that the characteristic chromatogram specifies the relative retention time of each characteristic peak, a flow rate of 0.80 ml / min is optimal.

[0103] Table 8. Results of relative retention time for different flow velocities

[0104]

[0105] Table 9. Results of relative peak area at different flow velocities

[0106]

[0107] Example 3: Investigation of column temperature

[0108] The difference between this embodiment and Example 1 is that the characteristic chromatograms of Jiaojian Shenqu formula granules (2307003S) were detected at different column temperatures in the chromatographic conditions. The column temperatures were set to 23℃, 25℃, and 27℃, respectively. The results are as follows: Figure 8-10 The table below shows that with fluctuations in column temperature, the separation effect of each chromatographic peak is good and the system adaptability parameters are relatively close. However, the relative retention time of each characteristic peak shows significant fluctuations. Considering that the characteristic chromatogram specifies the relative retention time of each characteristic peak, a column temperature of 25℃ is optimal.

[0109] Table 10. Results of relative retention times at different column temperatures

[0110]

[0111]

[0112] Table 11. Relative peak area at different column temperatures

[0113]

[0114] Example 4: Column Analysis

[0115] The difference between this embodiment and Example 1 is that different chromatographic columns were used to detect the characteristic chromatograms of Jiaojian Shenqu formula granules (2307003S). The selected columns were: Column 1: Inertil ODS-3; Column 2: Agilent SB-C18; Column 3: Waters HSS T3. The results are as follows: Figure 11-13 As shown in the table below, the results indicate that different chromatographic columns have a certain impact on the information content of chromatographic peaks and system adaptability parameters. After comparing and analyzing the results, Inertil ODS-3 showed better resolution and peak shape, therefore, Inertil ODS-3 column is the best choice.

[0116] Table 12. Comparison of relative retention times for different brands of chromatographic columns

[0117]

[0118]

[0119] Table 13 Comparison of relative peak areas of different brands of chromatographic columns

[0120]

[0121] Experimental Methodology Validation

[0122] 1. Instrument precision

[0123] The same Jiaojian Shenqu formula granules (2307003S) test solution was used and injected 6 times in accordance with the method in Example 1. The RSD of the relative retention time of the characteristic peak should be less than 2.0%. The results show that the instrument precision is good.

[0124] Table 14. Relative retention time results of precision test

[0125]

[0126]

[0127] Table 15. Results of relative peak area in precision test

[0128]

[0129]

[0130] 2. Repeatability experiment

[0131] Six test solutions were prepared in parallel according to the method in Example 1, and the RSD of the relative retention time of the characteristic peak was calculated to be less than 2.0%. The results showed that the method had good repeatability.

[0132] Table 16. Results of Relative Retention Times in Repeatability Tests

[0133]

[0134]

[0135] Table 17. Results of Relative Peak Area in Repeatability Tests

[0136]

[0137] 3. Stability test

[0138] The same Jiaojian Shenqu formula granules (2307003S) test solution was taken and measured at 0h, 2h, 4h, 8h, 12h and 24h according to the method of Example 1. The RSD of the relative retention time of the characteristic peak should be less than 2.0%. The results showed that the test solution was stable within 24h and met the measurement requirements.

[0139] Table 18. Results of relative retention times in stability tests

[0140]

[0141] Table 19. Results of relative peak area in stability test

[0142]

[0143]

[0144] 4. Exclusivity

[0145] Accurately pipette 10 μl each of the test solution, negative blank control solution (water), reference standard solution, and reference herbal medicine solution, and inject them separately into the high-performance liquid chromatograph. Perform the test according to the method in Example 1. The results are as follows: Figures 14-17 Negative results indicate no interference.

[0146] 5. Intermediate precision (personnel)

[0147] Three researchers prepared and measured the test solution of Jiaojian Shenqu formula granules (2307003S) according to the method in Example 1. The RSD of the relative retention time of the characteristic peak was calculated to be less than 2.0%, and the results showed that the intermediate precision was good.

[0148] Table 20. Intermediate Precision (Personnel) Relative Retention Time

[0149]

[0150]

[0151] Table 21. Relative Peak Area of ​​Intermediate Precision (Personnel)

[0152]

[0153] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting the characteristic chromatogram of Jiaojian Shenqu formula granules, characterized in that, The detection was performed using high-performance liquid chromatography (HPLC), and the chromatographic conditions were as follows: Octadecylsilane-bonded silica gel was used as the packing material; methanol was used as mobile phase A, and 0.2 wt% phosphoric acid solution was used as mobile phase B; the gradient elution program was as follows: For 0-15 min, the volume ratio of mobile phase A to mobile phase B is 0% : 100%. Over 15-50 minutes, the volume ratio of mobile phase A to mobile phase B changes from 0%:5% to 100%:95%. After 50-60 minutes, the volume ratio of mobile phase A to mobile phase B changes from 5%:95% to 8%:92%. The volume ratio of mobile phase A to mobile phase B is 8% : 92% for 60-65 minutes. Detection wavelength: 262nm; The preparation of the test solution includes: taking the test sample, adding solvent for extraction, filtering, and taking the filtrate to obtain the solution; the solvent is water. Preparation of reference solutions: Adenine, hypoxanthine, xanthine, uridine, and thymine were prepared into reference solutions respectively.

2. The method for detecting the characteristic spectrum of Jiaojian Shenqu formula granules according to claim 1, characterized in that, The chromatographic conditions include at least one of the following: 1) The flow rate is 0.75-0.85 mL / min; 2) Column temperature is 23-27℃; 3) The specifications of the chromatographic column are: column length 250mm, inner diameter 4.6mm, and particle size 5µm; 4) The injection volume is 5-15µL.

3. The method for detecting the characteristic spectrum of Jiaojian Shenqu formula granules according to any one of claims 1-2, characterized in that, The mass-to-volume ratio of the test sample to the solvent is (0.25-1):10, with a ratio of g / ml.

4. The method for detecting the characteristic spectrum of Jiaojian Shenqu formula granules according to any one of claims 1-2, characterized in that, It also includes the preparation of a reference solution for the control medicinal slices: take the Jiaojian Shenqu control medicinal slices, add solvent to extract, filter, and take the filtrate to obtain the solution.

5. The method for detecting the characteristic spectrum of Jiaojian Shenqu formula granules according to claim 4, characterized in that, The solvent is water; And / or, the mass-to-volume ratio of the Jiao Jian Shen Qu reference herbal slices to the solvent is (0.5-2):10-40, with a ratio of g / ml; And / or, take 0.5-2 parts by weight of Jiaojian Shenqu reference decoction pieces and add them to 10-40 parts by volume of water, heat and reflux to extract for 0.75-1.5 hours, filter, evaporate the filtrate to dryness, add the residue to 5-10 parts by volume of water, sonicate, filter, and take the filtrate.

6. The method for detecting the characteristic spectrum of Jiaojian Shenqu formula granules according to any one of claims 1-3, characterized in that, It also includes the preparation of reference solution: take an appropriate amount of uridine reference standard, weigh it accurately, and add water to prepare a solution containing 10-100 μg per 1 ml.

7. A method for detecting the content of Jiaojian Shenqu formula granules, characterized in that, The method includes high-performance liquid chromatography (HPLC) as used in the detection method for the characteristic chromatogram of Jiaojian Shenqu formula granules according to any one of claims 1-6.

8. The method for detecting the characteristic chromatogram of Jiaojian Shenqu formula granules according to any one of claims 1-6 or the method for determining the content of Jiaojian Shenqu formula granules according to claim 7, applied to the quality testing of Jiaojian Shenqu formula granules.

9. A method for quality testing of Jiaojian Shenqu formula granules, characterized in that, The method includes the steps of obtaining the characteristic spectrum of the test sample by the detection method of the characteristic spectrum of Jiaojian Shenqu formula granules according to any one of claims 1-6, and comparing the characteristic spectrum with the reference characteristic spectrum; The reference feature spectrum is obtained by fitting the feature spectrum obtained by using at least one batch of Jiaojian Shenqu formula granules as a standard according to the detection method of the feature spectrum of Jiaojian Shenqu formula granules as described in any one of claims 1-6.

10. The quality testing method for Jiaojian Shenqu formula granules according to claim 9, characterized in that, The reference characteristic spectrum includes 15 characteristic peaks, with peak 9 corresponding to uridine. Peak 9 is used as the reference peak S. The relative retention times of peaks 1-8 and peaks 10-15 should be within ±10% of the specified values. The specified values ​​for peak 1 are 0.16, peak 2 is 0.18, peak 3 is 0.21, peak 4 is 0.29, peak 5 is 0.34, peak 6 is 0.39, peak 7 is 0.44, peak 8 is 0.84, peak 10 is 1.08, peak 11 is 1.27, peak 12 is 1.43, peak 13 is 1.55, peak 14 is 1.87, and peak 15 is 2.

26.

11. The quality testing method for Jiaojian Shenqu formula granules according to claim 10, characterized in that, Peak 4 corresponds to adenine, peak 7 corresponds to hypoxanthine, peak 8 corresponds to xanthine, peak 9 corresponds to uridine, and peak 10 corresponds to thymine.

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